Cultured islet cluster allografts were not rejected when placed under the kidney capsule of immunocompetent allogeneic recipients differing from the donor at the major histocompatibility complex (MHC). Rejection occurred if the recipients were injected with T cells from donors syngeneic to the recipient who had been previously primed to lymphoid cells of the graft donor haplotype. T cells from normal, unprimed donors did not induce rejection. Elimination of the cytotoxic/suppressor subset of T cells abrogated rejection. One conclusion of this result is that cytotoxic T lymphocytes (CTL) are necessary for graft rejection in this animal model system. The aim of the project is to determine whether the CTL are sufficient or if collaboration with other T cell subsets is required. The hypothesis to be tested is that CTL specific for the class I MHC alloantigens of the cultured islet allograft require help, most likely in the form of IL-2, from helper T cells specific for class I MHC antigens (class I specific helpers) and shed graft alloantigens represented in the context of the graft recipients MHC (physiologic helper). In this proposal an in vivo separation procedure (negative selection) is described. With negative selection the data presented show that it should be possible to distinguish, by function, CTL from class I specific and physiologic helpers. The data presented also will show that elimination, via negative selection, of all three subsets abolishes the capacity of the remaining T cells to induce rejection. By appropriate selection procedures, described in detail in the text, it will be possible to determine if the CTL subset by itself or in collaboration with other T cell subsets can induce graft rejection. This study, which uniquely identifies T cell subsets by function and specificity, should provide useful information on the general question of the mechanism of T cell mediated graft rejection.